Comparison of five plating media for isolation of Salmonella species from human stools.

A comparative study was carried out to evaluate the performances of different culture media for the recovery of Salmonella spp. from 1,000 routine samples of human stools. By direct plating we tested Salmonella-Shigella agar (SS), Hektoen enteric agar (HE), bismuth sulfite agar (BS), novobiocin-brilliant green-glycerol-lactose agar (NBGL) and SM-ID medium (SM), and after selenite enrichment, we tested all of the media except HE. C8-esterase and oxidase tests were used for the screening of Salmonella spp. on SS and HE. The total number of Salmonella isolates from direct culture was 74, with respective sensitivities and positive predictive values (PPVs) of 78.4 and 61%, 64.9 and 18.7%, 36.5 and 34.2%, 55.4 and 20.7%, and 39.2 and 43.9% for NBGL, SS, HE, BS, and SMID, respectively. After enrichment, the total number of Salmonella isolates was 88. The respective sensitivities and PPVs obtained were 90.9 and 62.5%, 92 and 17%, 90.9 and 32% and 93.2 and 71.3% for NBGL, SS, BS, and SM, respectively. According to our results, NBGL in direct plating was the medium with the highest sensitivity with respect to the sensitivities of the other media, with significant statistical differences (P < 0.05). Likewise, the PPV for NBGL was also the highest (61%). After enrichment in selenite broth, the sensitivities of the four media tested were similar, with the best PPV obtained with SM (71.3%); this was followed by NBGL (62.5%). When C8-esterase was used on SS or HE, the PPVs improved from less than 40% to about 100%.     

Comparison of media for screening of diarrheic stools for the recovery of Clostridium difficile.

Recoveries of Clostridium difficile from stool specimens by using three media, cycloserine-mannitol agar (M-CMA), cycloserine-mannitol-blood agar (M-CMBA), and cycloserine-cefoxitin agar (M-CCA), were compared. Of 321 clinical specimens, 37 yielded C. difficile. Thirty-four were positive on M-CCA, 21 were positive on M-CMA, and 20 were positive on M-CMBA. M-CCA recovered significantly more C. difficile than did M-CMBA or M-CMA.     

Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin.

Stools from patients with antibiotic-associated diarrhea or colitis were cultured to detect the presence of Clostridium difficile. All specimens contained a cytotoxin which was neutralized by Clostridium sordellii antitoxin. Initial testing employed several methods with comparative merits in recovering this organism. These included the use of nonselective media, antibiotic-incorporated media, alcohol shock, and paracresol-containing broth. Optimal results were achieved with primary plating of serial dilutions onto a selective agar containing cycloserine and cefoxitin. This technique was then employed in a large number of specimens. The overall results showed that C. difficile was recovered in specimens from 71 of 73 patients. All isolates of C. difficile produced a cytotoxin which was neutralized by C. sordellii antitoxin in vitro. These results verify the utility of this medium and support the concept that C. difficile accounts for the cytotoxin found in stools in nearly all cases.     

Simplified procedure for the routine isolation of Clostridium difficile from faeces.

The use of alcohol, at a final concentration of 50%, as a selective procedure for the isolation of Clostridium difficile was compared to a selective medium containing 250 microgram /ml of cycloserine and 10 microgram /ml of cefoxitin. Of 266 faecal samples 82 were shown to be positive by one or other method. Seventy-seven (94%) of these were detected by the selective agar (SA) and 72 (88%) by the alcohol procedure (AP). Ten samples (12%) were positive only by SA and five samples (6%) by AP only. The AP was further modified so that all manipulations prior to incubation were performed on the open bench. Of 18 positive samples, 18 (100%) were detected by SA and 16 (89%) by AP.     

Selective and differential medium for isolation of Clostridium difficile.

Clostridium difficile is a recognized cause of pseudomembranous (antimicrobial agent-associated) colitis and may be one of the causes of antimicrobial agent-induced diarrhea. A selective and differential agar medium that contains cycloserine, cefoxitin, fructose, and egg yolk (CCFA) was developed to facilitate the isolation of C. difficile from fecal specimens. Quantitative cultures of 16 stock strains of C. difficile on this medium (and on a medium containing cycloserine, fructose, and egg yolk) yielded counts equivalent to those obtained on blood agar; other media selective for clostridia, including Clostrisel agar, reinforced clostridial agar plus 0.2% para-cresol, and egg yolk-neomycin agar (the latter was inoculated with cultures subjected to prior heat shocking), were also tested and found to be inhibitory to the growth of C. difficile. Of 28 fecal or colostomy effluent specimens cultured on the above media, 14 yielded C. difficile. CCFA was found to be the most sensitive and selective of these media for the recovery of C. difficile. Colonies of C. difficile growing on CCFA had distinctive morphological and fluorescent properties which were sufficient for presumptive identification. CCFA should provide a rapid method for the screening of fecal specimens from patients with antimicrobial agent-associated diarrhea or colitis for C. difficile.     

Comparison of typing methods for Clostridium difficile isolates.

A simple discriminative typing method for Clostridium difficile has been developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and restriction enzyme analysis are relatively simple techniques but are difficult to evaluate, especially the restriction enzyme analysis. Immunoblotting and restriction fragment length polymorphism typing facilitate simple discrimination of patterns.     

Use of gas-liquid chromatography as a screening test for toxigenic Clostridium difficile in diarrhoeal stools

In order to determine if gas-liquid chromatography (GLC) on concentrated stool extracts could be substituted to cell culture assay for cytotoxicity, we prospectively studied 154 diarrhoeal stools submitted for detection of Clostridium difficile toxin. Isocaproic-positive samples were cultured on egg yolk agar supplemented with cycloserine, cefoxitin and fructose for isolation of C difficile, and on egg yolk agar plus kanamycin for isolation of other clostridium species. Of the 154 samples, 129 were GLC-negative (height of the isocaproic peak less than 1.2 cm) and were toxin-negative. Twenty-five stools yielded isocaproic acid; C difficile isolated from 13 of them, six of which were also toxin-positive. Four other isocaproic-positive samples yielded C bifermentans and C sordellii; all were toxin-negative. These results indicate that a negative GLC is an excellent screening test for excluding C difficile infection; positive results must be checked by toxin testing and culture since they are not necessarily associated with the presence of C difficile or its toxin.     

Comparison of two toxins produced by Clostridium difficile.

Clostridium difficile was shown to produce a toxin which could be biochemically separated from the previously described cytotoxin of the same organism. The two proteins differ in biological activity and physical properties. Antiserum prepared to the second toxin does not neutralize the biological activity of the cytotoxin, and immunological cross-reactivity could not be demonstrated. However, some relationship may exist between the two toxins, since the newly described toxin degrades on polyacrylamide electrophoresis into two molecules, one of which appears to migrate with the band of purified cytotoxin. We suggest that this newly described toxin be designated toxin A until its primary biological activity and physical relationship to cytotoxin is determined. This toxin is active in biological assays of enteric disease and may play an important role in C. difficile-induced colitis.     

Comparison of methods for recovery of Clostridium difficile from an environmental surface

Survival of Clostridium difficile in an aerobic environment is possible because of spore formation. When sodium taurocholate is substituted for the egg yolk of a selective medium, cycloserine-cefoxitin-fructose-agar (CCFA), enhanced recovery of C. difficile spores is shown. This selective medium (TCCFA) does not improve recovery of vegetative forms. In this study, dry and saline-moistened swabs, adhesive paddles, and Rodac plates containing CCFA and TCCFA were compared in their ability to recover C. difficile spores from an inoculated surface. Rodac plates grew 20 to 25 times as many spores on TCCFA as on CCFA. Saline-moistened swabs recovered fewer organisms than Rodac plates. Dry swabs and adhesive paddles rarely recovered spores. Prereduction of agar in an anaerobic chamber was not necessary for optimal spore recovery. Optimal growth of vegetative C. difficile required prereduced media. Agar prereduced for 2 h supported the growth of 12 C. difficile isolates as well as agar prereduced for 18 h. Vegetative cells of C. difficile survived for only 15 min in room air. Use of Rodac plates containing TCCFA is preferred for detection of C. difficile spores in the hospital environment.     

Clostridium difficile plasmid isolation as an epidemiologic tool

A large hospital outbreak ofClostridium difficile diarrhea at the Minneapolis Veterans Administration Medical Center (MVAMC) was studied by plasmid profile typing. Plasmids were obtained from 30 (37 %) of 82 clinical isolates from MVAMC patients and 10 (67 %) of 15 non-MVAMC isolates. While bacteriophage plus bacteriocin typing and polyacrylamide gel electrophoresis (PAGE) plus bacterial agglutination typing proved more universally applicable, plasmid profiles may be useful for tracing isolated epidemic outbreaks, reinfections and relapses caused by plasmid-bearing strains.     

Comparison of strain typing results for Clostridium difficile isolates from North America

Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.     

Comparison of serogrouping and polyacrylamide gel electrophoresis for typing Clostridium difficile.

A typing scheme for Clostridium difficile based on slide agglutination with rabbit antisera was previously described. It allows the differentiation of 10 serogroups designated A, B, C, D, F, G, H, I, K, and X. We studied the correlation between serogrouping and polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins. A total of 202 isolates from different sources were analyzed by PAGE after ultrasonic disintegration of cells from an 18-h liquid culture and treatment with sodium dodecyl sulfate and 2-mercaptoethanol. A total of 21 different patterns were observed. The reference strains from the 10 serogroups showed different profiles. For each serogroup except A, the patterns obtained with the clinical isolates were identical to the patterns obtained with the reference strains. For the 48 strains belonging to serogroup A, 12 different profiles were observed. Five of these involved strains isolated from patients with antibiotic-associated diarrhea. Typing by sodium dodecyl sulfate-PAGE thus correlates with serogrouping. In addition, it allows discrimination within the heterogeneous serogroup A.     

Clostridium difficile and its cytotoxin in the stools of young hospitalized children. Influence of antibiotic treatment

Clostridium difficile has been searched in 153 stool samples from 138 children aged 0 to 12 months. We divided the population in two groups depending on the antibiotic treatment. We have found C difficile in 39 samples (25%). The colonization rate increases with age ranging from 5% before 1 month, to 36% between 1 and 6 months and 54% between 6 and 13 months. An environmental sampling yielded once C difficile. Contamination may be related to the environment. 29% of the isolates produced a cytopathic toxin. Toxin titers in infants' stools range from 1/160 to 1/10240. One only of these children had diarrhea. C difficile and its toxin does not seem to infer any signs of enteric illness with infants. The results obtained with the group of non treated infants are not significantly different from the ones of the other group: the colonization rates are 21% in the non treated group and 29% in the other group. The rate of strains yielding a cytophatic toxin is similar in the 2 groups. It seems reasonable to agree that antibiotics do not influence the settlement of C difficile in infants' intestine.     

Clostridium difficile and its cytotoxin in diarrhoeic stools of hospitalized patients. Toxigenic potential of the isolates

Cytotoxin assay and culture for Clostridium difficile were performed on 303 diarrhoeic stools from 261 hospitalized patients. Specimens from 42 patients were positive by at least one of the methods, and 40 of them had an antibiotic-associated diarrhoea. The cytotoxin assay was positive in 5 of 7 patients with pseudomembranous colitis. Thirteen had an appropriate response to specific therapy and the remainder have resolved of diarrhoea without C. difficile directed chemotherapy. These findings show the lack of reliability of the cytotoxin assay for the diagnosis of C. difficile antibiotic-associated diarrhoea. The 6 strains isolated from patients with pseudomembranous colitis were examined for enterotoxin by the rabbit ileal loop test: 4 produced both toxins, 2 only enterotoxin. Both toxins could therefore not be essential for the clinical expression of the disease.     

Differential and selective medium for isolation of Yersinia enterocolitica from stools.

A new differential and selective medium, DYS agar, was developed and evaluated from the isolation of Yersinia enterocolitica. Ther bile salts content of the medium resulted in high selectivity, and inclusion of arabinose, lysine, and arginine rendered Y. Enterocolitica very distinct from Proteus spp., Pseudomonas spp., and other members of the family Enterobacteriaceae. DYS medium was more efficient for the isolation of Y. enterocolitica from experimentally inoculated fecal specimens than MacConkey, deoxycholate-citrate, and salmonella-shigella agars. Although the medium showed selectivity similar to that of another relatively new medium. Y medium (a selective medium for Y. enterocolitica containing sodium oxalate). DYS agar was found to be superior to Y medium in terms of differentiation of Y. enterocolitica from other intestinal organisms. DYS medium is simple to prepare.